Bozza P T, Payne J L, Goulet J L, Weller P F
Harvard Thorndike Laboratory, Beth Israel Hospital, Boston, Massachusetts 02215, USA.
J Exp Med. 1996 Apr 1;183(4):1515-25. doi: 10.1084/jem.183.4.1515.
Lipid bodies, lipid rich cytoplasmic inclusions, are characteristically abundant in vivo in leukocytes associated with inflammation. Because lipid bodies are potential reservoirs of esterified arachidonate and sites at which eicosanoid-forming enzymes may localize, we evaluated mechanisms of lipid body formation in neutrophils (PMN). Among receptor-mediated agonists, platelet activating factor (PAF), but not C5a, formyl-methyl-phenylalanine, interleukin 8, or leukotriene (LT) B4, induced the rapid formation of lipid bodies in PMN. This action of PAF was receptor mediated, as it was dose dependently inhibited by the PAF receptor antagonist WEB 2086 and blocked by pertussis toxin. Lipid body induction by PAF required 5-lipoxygenase (LO) activity and was inhibited by the 5-lipoxygenase-activating protein antagonist MK 886 and the 5-LO inhibitor zileuton, but not by cyclooxygenase inhibitors. Corroborating the dependency of PAF-induced lipid body formation on 5-LO, PMN and macrophages from wild-type mice, but not from 5-LO genetically deficient mice, formed lipid bodies on exposure to PAF both in vitro and in vivo within the pleural cavity. The 5-LO product inducing lipid body formation was not LTB4 but was 5(S)-hydroxyeicosatetraenoic acid [5(S)-HETE], which was active at 10-fold lower concentrations than PAF and was also inhibited by pertussis toxin but not by zileuton or WEB 2086. Furthermore, 5-HETE was equally effective in inducing lipid body formation in both wild-type and 5-LO genetically deficient mice. Both PAF- and 5(S)-HETE-induced lipid body formation were inhibited by protein kinase C (PKC) inhibitors staurosporine and chelerythrine, the phospholipase C (PLC) inhibitors D609 and U-73122, and by actinomycin D and cycloheximide. Prior stimulation of human PMN with PAF to form lipid bodies enhanced eicosanoid production in response to submaximal stimulation with the calcium ionophore A23187; and the levels of both prostaglandin (PG) E2 and LTB4 correlated with the number of lipid bodies. Furthermore, pretreatment of cells with actinomycin D or cycloheximide inhibited not only the induction of lipid body formation by PAF, but also the PAF-induced "priming" for enhanced PGE2 and LTB4 in PMN. Thus, the compartmentalization of lipids to form lipid bodies in PMN is dependent on specific cellular responses that can be PAF receptor mediated, involves signaling through 5-LO to form 5-HETE and then through PKC and PLC, and requires new protein synthesis. Since increases in lipid body numbers correlated with priming for enhanced PGE2 and LTB4 production in PMN, the induction of lipid bodies may have a role in the formation of eicosanoid mediators by leukocytes involved in inflammation.
脂质体是富含脂质的细胞质内含物，其在体内与炎症相关的白细胞中通常大量存在。由于脂质体是酯化花生四烯酸的潜在储存库，也是类二十烷酸形成酶可能定位的位点，我们评估了中性粒细胞（PMN）中脂质体形成的机制。在受体介导的激动剂中，血小板活化因子（PAF）而非C5a、甲酰甲硫氨酸、白细胞介素8或白三烯（LT）B4能诱导PMN中脂质体的快速形成。PAF的这一作用是受体介导的，因为它被PAF受体拮抗剂WEB 2086剂量依赖性抑制，并被百日咳毒素阻断。PAF诱导脂质体形成需要5-脂氧合酶（LO）活性，并被5-脂氧合酶激活蛋白拮抗剂MK 886和5-LO抑制剂齐留通抑制，但不被环氧化酶抑制剂抑制。证实PAF诱导脂质体形成对5-LO的依赖性的是，野生型小鼠而非5-LO基因缺陷小鼠的PMN和巨噬细胞，在体外和胸腔内体内暴露于PAF时均形成脂质体。诱导脂质体形成的5-LO产物不是LTB4，而是5（S）-羟基二十碳四烯酸[5（S）-HETE]，其活性浓度比PAF低10倍，也被百日咳毒素抑制，但不被齐留通或WEB 2086抑制。此外，5-HETE在诱导野生型和5-LO基因缺陷小鼠的脂质体形成方面同样有效。PAF和5（S）-HETE诱导的脂质体形成均被蛋白激酶C（PKC）抑制剂星形孢菌素和白屈菜红碱、磷脂酶C（PLC）抑制剂D609和U-73122以及放线菌素D和放线菌酮抑制。预先用PAF刺激人PMN形成脂质体可增强对钙离子载体A23187亚最大刺激的类二十烷酸产生；前列腺素（PG）E2和LTB4的水平均与脂质体数量相关。此外，用放线菌素D或放线菌酮预处理细胞不仅抑制PAF诱导的脂质体形成，还抑制PAF诱导的PMN中PGE2和LTB4增强的“启动”作用。因此，PMN中脂质形成脂质体的区室化依赖于可由PAF受体介导且涉及通过5-LO形成5-HETE然后通过PKC和PLC的信号传导的特定细胞反应，并且需要新的蛋白质合成。由于脂质体数量的增加与PMN中PGE2和LTB4产生增强的启动相关，脂质体的诱导可能在参与炎症的白细胞形成类二十烷酸介质中起作用。
